Skeletal myogenesis during both embryonic development and adult muscle regeneration is a complex process mediated by a myriad of signaling pathways and transcriptional events. Myogenic precursor cells and satellite cells either proliferate or differentiate based on cues from mitogen-activated protein kinases (MAPKs). The Siderovski laboratory recently discovered a novel pro-neuritogenesis role for the GPCR negative regulator RGS12 in PCI2 cells and primary neurons in its function as a scaffold for H-Ras, B-Raf, and the complete extracellular-regulated kinase (ERK) MAPK cascade. During mouse development, RGS12 expression is seen restricted to specific neuronal populations and skeletal muscle. In a skeletal myoblast/satellite cell model (C2C12 cells), H-Ras and ERK1/2 activation promote proliferation and inhibit myotube formation, whereas rapid inactivation of ERK1/2 is necessary for myogenesis to occur. RGS12 is expressed in C2C12 cells and primary myoblasts. Interestingly, during C2C12 differentiation, RGS12 levels are down-regulated. We hypothesize that RGS12 coordinates H-Ras/ERK MAPK signaling in myoblasts and satellite cells to promote proliferation and inhibit differentiation. Loss of RGS12 would disrupt this scaffolding function, allowing myogenesis to proceed. We will test this hypothesis with cell biological studies, including siRNA depletion of RGS12 and suspected regulators of its stability, as well as chemical biology studies designed to identify and validate small molecule inhibitors of RGS12 cellular action. Aim 1: Determine the role of the RGS12-assembled H-Ras/MAPK signaling complex in H-Ras-mediated suppression of myogenic differentiation Aim 2; Determine mechanism and significance of RGS12 degradation during myogenic differentiation Aim 3: Identify and validate small molecule inhibitors of RGS12 action as potential agents in modulating myogenic differentiation Our results from studying RGS12 function and its inhibition will have important implications for adult skeletal muscle regeneration, where a delicate balance between proliferating and differentiating satellite cells is required to restore physiologically functional skeletal muscle after damage and during aging. We also hope to highlight the RGS12 scaffold as a point of therapeutic intervention in metastatic rhabdomyosarcoma, a satellite cell-derived cancer where H-Ras mutations and increased ERK activity have been described.